Construction and application of recombinant PRRSV strain for expressing porcine circovirus Cap protein

Abstract

The invention discloses construction and application of a recombinant PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) strain for expressing porcine circovirus Cap protein. The recombinant PRRSV capable of expressing the Cap protein of the porcine circovirus type 2 is successfully constructed and saved by adopting a reverse genetic manipulation technology. The modified virus is inoculated to Marc-145 cells for subculture, and the result shows that the modified virus can trigger cytopathy and stably express Cap protein. The obtained recombinant strain shows a stable passage capability in a Marc-145 cell, and has an extremely high virus load. The strain can be used as a vaccine candidate strain, provides multiple protection for PRRSV and porcine circovirus of different pedigree, and provides a brand new solution for healthy breeding of pigs.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and in particular relates to the construction and application of a recombinant PRRSV strain expressing porcine circovirus Cap protein. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious diseases in the global swine industry, causing significant economic losses annually. PRRS is caused by porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae. PRRSV is divided into two major types: PRRSV-1 and PRRSV-2. PRRSV-2 is predominantly prevalent in my country, with the main subtypes including lineages 1 (NADC30-like), 3 (QYYZ-like), 5 (VR2332-like), and 8 (HP-PRRSV vaccine-derived strain). Currently, most clinical cases detected in the field are primarily mixed infections of lineages 1 and 8, which increases the technical complexity of on-site prevention and control. Furthermore, although multiple PRRSV vaccines are currently in use, their effectiveness is limited due to the rapid mutation of PRRSV, especially against new lineage 1 (NADC30-like) PRRSV variants. Therefore, the development of novel, broad-spectrum vaccines and prevention and control strategies is crucial for effectively controlling the prevalence of PRRS.

[0003] Porcine circovirus (PCV) is a major pathogen in the global swine industry, with PCV2 being the primary cause of porcine circovirus disease (PCVD). The clinical manifestations of PCVD are complex and diverse, encompassing porcine circovirus-associated disease (PCVAD), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and congenital tremor. PCV2 infection not only severely impacts pig health and production performance, but also, when co-infected with PRRSV, can lead to even more severe clinical symptoms and pathological changes, further exacerbating the economic burden on the swine industry.

[0004] Given the profound impact of PRRS and PCVD on the swine industry, particularly the severe challenges posed by co-infections of PRRSV and PCV2, the development of novel vaccines or control strategies that simultaneously target both pathogens is urgent. This present invention, based on this background, aims to construct a recombinant PRRSV strain expressing the porcine circovirus Cap protein, with the goal of demonstrating significant advantages in preventing and controlling co-infections of PRRS and PCV, reducing stress responses in animals caused by multiple injections, minimizing economic losses, and achieving the goal of providing multiple vaccinations with a single injection.

[0005] The present invention successfully constructed a recombinant strain by embedding the Cap protein from PCV2 into a reverse genetic vector of the TJ1501 virus. This strain is expected to become a potential vaccine candidate, capable of providing multiple protection against different lineages of PRRSV and porcine circovirus type 2. Summary of the Invention

[0006] In view of this, the present invention relates to the construction and application of a recombinant PRRSV strain expressing porcine circovirus Cap protein.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: A recombinant PRRSV strain expressing the porcine circovirus Cap protein is named FX1701-CAP strain. Its main characteristic is that the constructed strain contains HP-PRRSV (30 amino acid deletion) and NADC30-LikePRRSV (131 amino acid deletion) as well as Cap protein.

[0008] The present invention further discloses a method for constructing a recombinant PRRSV strain expressing porcine circovirus Cap protein, the steps of which are as follows: (1) Extract the PRRSV 1501 strain virus liquid, obtain the full-length viral cDNA by reverse transcription, and use specific primers for PCR amplification to obtain the gene fragment of the 1501 strain; (2) Treat the gene fragment plasmid with PacⅠ, BstBI, AfⅡ, AscⅠ, and MluⅠ endonucleases, connect the gene fragments into a full-length genomic fragment through enzyme digestion and homologous recombination, and insert it into the PSKⅡ vector; (3) In a water bath, verify the plasmid using EcoRI single enzyme digestion and perform full-length verification of the plasmid to ensure the correct length of the plasmid; (4) Use specific primers to amplify the Cap protein gene in porcine circovirus and insert it between ORF1a and ORF2b of the PRRSV reverse genetic vector by homologous recombination to obtain a recombinant plasmid. The length and correctness of the inserted sequence are verified by enzyme digestion. The primer sequences used in steps (1) to (4) are as follows:

[0009] The present invention further discloses the use of a recombinant PRRSV strain expressing the porcine circovirus Cap protein in the preparation of a vaccine that provides multiple protection against different lineages of PRRSV and porcine circovirus type 2. This multiple protection is intended to provide a new candidate vaccine strain for the prevention and control of mixed infections of different lineages of PRRS and PCV2. Experimental results show that this recombinant vaccine strain is effective against different lineages of PRRSV and PCV2, providing excellent immune protection in piglets.

[0010] The construction and application of the recombinant PRRSV strain expressing the porcine circovirus Cap protein disclosed in the present invention have the following positive effects compared with the existing technology: (1) It can provide piglets with immune protection against PRRSV and PCV2 infection at the same time; (2) Demonstrated significant advantages in preventing and controlling mixed infections of different lineages of PRRS and PCV2; (3) Reduces stress and other problems caused by multiple vaccinations to piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A schematic diagram of the plasmid map of the PRRSV reverse genetic vector platform constructed in Example 1 is shown; Figure 2 The gel electrophoresis results of double enzyme digestion verification of the PRRSV reverse genetic vector plasmid constructed in Example 1 are presented; Figure 3 The figure shows the pathological changes induced by the reverse genetic vector in Example 1 in Marc-145 cells; Figure 4 The growth curves of the reverse genetic vector and the original strain in implementation 1 were compared; Figure 5 Schematic diagram of the reverse genetic vector plasmid map of the PRRSV recombinant cap protein in implementation 2; Figure 6 A schematic diagram of gel electrophoresis of the amplified cap protein in Example 2 is shown; Figure 7 The figure shows the pathological changes induced by the recombinant virus in Example 2 in Marc-145 cells; Figure 8 This is a WB image for the cap protein expression verification in implementation 2; Figure 9 ELISA levels of protection after immunization in step 3 are shown. DETAILED DESCRIPTION

[0012] The present invention is described in detail below by specific embodiments. Unless otherwise specified, the technical means adopted in the present invention are conventional methods well known to those skilled in the art. In addition, the embodiments provided should be regarded as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are defined only by the claims. For those skilled in the art, various adjustments or modifications of the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0013] The conventional experimental methods in the following examples are based on the third edition of Molecular Cloning Laboratory Manual compiled by Sambrook et al. (Beijing: Science Press, 2002). The use of instruments is based on the corresponding instrument operating instructions.

[0014] In the examples herein, the DH5α competent cells used were purchased from Sangon Biotech (Shanghai) Co., Ltd., and the viruses were rescued to obtain the FX1701 strain (known) and the FX1701-CAP strain (the strain obtained in the present invention). Other cells used included the BHK-21 cell line, the Marc-145 cell line, and primary alveolar macrophages (PAMs) (commercially available).

[0015] In the examples of the present invention, other reagents used include: RNase Free H2O and trypsin cell digestion solution (phenol red) were purchased from Solarbio; TRIpure Reagent total RNA extraction reagent was purchased from Adlai Biotechnology; 2× PrimeSTAR MAX DNA Polymerase, T4 ligase, and T cloning kit were purchased from TAKARA; One StepClone Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; DMEM was purchased from HyClone Biochemical Products Co., Ltd.; fetal bovine serum was purchased from Sigma; DNA restriction endonucleases and LipofectamineTM3000 Transfection Reagent were purchased from Thermo Fisher. Example 1

[0016] Based on the known TJ1501 strain, the reverse genetic vector strain FX1701 adapted to in vitro passage in Marc-145 cells was successfully obtained.

[0017] 1.1 Schematic diagram of the construction of FX1701 reverse genetic vector using Snapgene software. Figure 1 .

[0018] 1.2 Primer design for construction of FX1701 reverse genetic vector Using Snapgene software, the TJ1501 strain was divided into four parts for amplification according to the simulated plasmid schematic diagram, and the corresponding primers were designed (see Table 1).

[0019]

[0020] 1.3 Reverse genetic vector construction methods First, fragments 1501-A, 1501-B, 1501-C, and 1501-D were amplified by PCR to obtain the full-length sequence of the original virus. Subsequently, the amplified fragments were ligated into the PSKⅡ vector using homologous recombination technology.

[0021] 1.3.1 Target fragment PCR amplification method and system In the present invention, amplification of target fragments is performed using PCR technology. The specific system and procedure of PCR amplification are detailed in Tables 2 and 3. The commercial kit used is 2× PrimeSTAR MAX DNA Polymerase, purchased from TAKARA.

[0022]

[0023] Using the above method, we can obtain Figure 1 The fragments of the original strain full-length sequence were spliced ​​together as shown in Figure 1. Separation was performed by 1% wide-pore gel electrophoresis for 35 minutes. Enzyme-cleaved bands were separated by agarose gel electrophoresis, and the target bands were subsequently excised and purified by gel extraction. The amplified target fragments were stored at -20°C.

[0024] 1.3.2 Vector Enzyme Digestion and Homologous Recombination Methods and Systems In the present invention, all vector enzyme digestion and homologous recombination methods and systems are carried out according to the following steps: First, the PSKⅡ vector is double-digested with PacⅠ and MluⅠ to prepare a linearized vector. The specific system is detailed in Table 4.

[0025]

[0026] According to the above system, the sample was digested at 37°C for 1 hour. The resulting bands were then separated by agarose gel electrophoresis, and the target band was excised and purified. This yielded the linearized PSKII vector, which was measured to have a concentration of 100 ng / μl and stored at -20°C.

[0027] Linearized fragments 1501-A, 1501-B, 1501-C, and 1501-D were homologously recombined with the linearized vector PSKⅡ using the One Step Clone Kit. The specific experimental system is detailed in Table 5.

[0028]

[0029] After the system reacts at 50°C for 30 minutes, immediately place on ice. Inoculate the transformed product into DH5α and select independent colonies for pure culture. Perform PCR on the bacterial solution using universal primers. Select PCR-positive colonies for overnight propagation, extract the plasmid, and verify it by double enzyme digestion with AscⅠ and BSTbⅠ. Subsequently, analyze the plasmid by 1% agarose gel electrophoresis to confirm the correct enzyme digestion bands. After that, store the correct plasmid at -20°C.

[0030] 1.4 Rescue of MARC-145 cells with reverse genetic vectors First, BHK-21 cells were seeded at a density of 6 × 10^5 cells / well in a 6-well cell culture plate using DMEM medium supplemented with 10% FBS and cultured in a 37°C, 5% CO2 incubator until the cell density reached approximately 80%. Cell transfection was performed according to the instructions for Lipofectamine™ 3000 Transfection Reagent.

[0031] The transfection method is as follows: Mix 2 μg of plasmid, 4 μL of P3000 plasmid, and 50 μL of Opti-MEM reagent to form Solution A. Then, mix 3 μL of Lip3000 and 50 μL of DMEM to form Solution B. After mixing Solution A and Solution B, let them stand at 25°C for 15 minutes before adding them to BHK-21 cells. See Table 6 for specific transfection systems.

[0032] 48 hours after cell transfection, the 6-well plate was sealed and frozen at -80°C. After repeated freezing and thawing twice, all the cell suspensions were collected and centrifuged at 12,000 × g for 5 minutes to collect the transfection supernatant.

[0033]

[0034] MARC-145 cells were seeded into 6-well cell culture plates at a density of 6 × 10^5 cells / well using 2 mL of DMEM medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. After the MARC-145 cells adhered, 500 μL of the transfection supernatant was overlaid on the MARC-145 cells. After incubation for 1 hour, the supernatant was discarded and DMEM medium containing 2% FBS was added to continue the culture.

[0035] The rescue results of MARC-145 cells are shown in Figure 3 , observation of lesions proves successful rescue.

[0036] 1.6 Determination of growth characteristics of strains adapted to MARC-145 cell culture First, the TCID50 titer of the fifth-generation reverse genetic vector was determined. The TCID50 assay was performed as follows: MARC-145 cells were seeded at a density of 5 × 10^4 cells / well in DMEM medium supplemented with 10% FBS in a 96-well cell culture plate. The cells were cultured in a 37°C, 5% CO2 incubator. When the cell density reached approximately 80%, the medium was switched to DMEM supplemented with 2% FBS. The viral stock solution was diluted 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, and 1,000,000-fold, respectively. Each dilution series was inoculated in triplicate wells of a 96-well plate. After adsorption for 1 hour, the medium was replaced with 200 μL of fresh DMEM supplemented with 2% FBS. After 72 hours, the number of wells with lesions was counted, and the TCID50 viral titer of the reverse genetic vector was calculated.

[0037] The growth of the reverse genetic vector on MARC-145 cells was evaluated by drawing a multi-step growth curve. The specific method is as follows: each virus liquid was inoculated into MARC-145 cells in a 6-well plate at an infection dose of 0.01 MOI. The supernatant was collected at 5 time points, namely 12 hours, 24 hours, 36 hours, 48 ​​hours and 72 hours, and frozen in a -80°C refrigerator. After all time points were collected, the TCID50 titer of the supernatant at different time points was determined and a standard curve was drawn. The results of the multi-step growth curve determination are shown in Figure 2. Figure 4 As shown, both the reverse genetic vector and the wild-type strain can be propagated on MARC-145 cells. Example 2

[0038] Based on the FX1501 reverse genetic vector, the recombinant vaccine candidate strain FX1701-CAP was obtained by in vitro passage of Marc-145 cells.

[0039] 2.1 Schematic diagram of the construction of recombinant plasmids using Snapgene software Figure 5 .

[0040] 2.2 Primer design for constructing recombinant plasmids Using Snapgene software, according to the schematic diagram of the simulated plasmid, the recombinant protein was divided into three parts for amplification, and the corresponding primers were designed (Table 7).

[0041]

[0042] 2.3 Methods for constructing recombinant plasmids First, PF, CAP, and PR fragments were amplified by PCR to obtain the full-length protein, and then these fragments were ligated into the FX1701 vector by homologous recombination.

[0043] 2.3.1 Target fragment PCR amplification method and system In the present invention, the target fragments were amplified using PCR amplification. The specific system and procedure of PCR amplification are detailed in Tables 8 and 9. The commercial kit used was 2× PrimeSTAR MAX DNA Polymerase, purchased from TAKARA.

[0044]

[0045] Using the above method, we can obtain Figure 5 Various fragments of the CAP protein sequence were detected. Enzyme-cleaved bands were separated by agarose gel electrophoresis using a wide-pore 1% gel for 35 minutes. The target bands were then excised and purified by gel extraction. The amplified target fragments were stored at -20°C.

[0046] 2.3.2 Vector Enzyme Digestion and Homologous Recombination Methods and Systems In the present invention, all vector digestion and homologous recombination methods and systems are carried out according to the following steps. First, the reverse genetics vector is double-digested with ASCII and ECORV to prepare a linearized vector. The specific double-digestion system is detailed in Table 10.

[0047]

[0048] According to the above system, the sample was digested at 37°C for 1 hour. The digested bands were then separated by agarose gel electrophoresis, and the target band was excised and purified by gel extraction. The linearized reverse genetic vector was obtained, with a concentration of 200 ng / μl, and stored at -20°C.

[0049] The linearized fragments PF, CAP, and PR were homologously recombined with the linearized vector using the One-Step Clone Kit. The specific experimental system is detailed in Table 11.

[0050]

[0051] Incubate the system at 50°C for 30 minutes and immediately place on ice. Transform the system into DH5α and isolate individual colonies for pure culture. Perform PCR on the bacterial suspension using universal primers. Select PCR-positive bacteria for overnight propagation, extract the plasmid, and verify by double enzyme digestion. Then, perform electrophoresis on a 1% agarose gel to confirm the correct enzyme digestion bands. Store the correct plasmid at -20°C.

[0052] 2.4 Rescue of MARC-145 cells with reverse genetic vectors First, BHK-21 cells were seeded at a density of 6 × 10^5 cells / well in a 6-well cell culture plate using DMEM medium supplemented with 10% FBS. The cells were cultured in a 37°C, 5% CO2 incubator until the cell density reached approximately 80%. Cell transfection was performed according to the instructions for Lipofectamine™ 3000 Transfection Reagent.

[0053] The transfection method is as follows: Mix 2 μg of plasmid, 4 μL of P3000 plasmid, and 50 μL of OPTI-MEM reagent to form Solution A. Then, mix 3 μL of Lipofectamine™ 3000 and 50 μL of DMEM to form Solution B. Then, mix Solution A and Solution B and incubate at 25°C for 15 minutes. Finally, add the mixture to BHK-21 cells. See Table 12 for specific transfection systems.

[0054] 48 hours after cell transfection, the 6-well plate was sealed and frozen at -80°C. After repeated freezing and thawing twice, the entire cell suspension was taken and centrifuged at 12,000 × g for 5 minutes to collect the transfection supernatant.

[0055]

[0056] Pre-seed MARC-145 cells at 6 × 10^5 cells / well in 2 mL of DMEM medium containing 10% FBS into a 6-well cell culture plate and incubate at 37°C, 5% CO2. After the MARC-145 cells adhere, overlay 500 μL of the transfection supernatant on the MARC-145 cells. After incubation for 1 hour, discard the supernatant and add DMEM medium containing 2% FBS to continue culturing.

[0057] The recombinant virus was serially passaged, and the pathological changes were shown in Figure 7. After stable inheritance, WB verification was performed using a monoclonal antibody against the Cap protein, and the WB band pattern was shown in Figure 7. Figure 8 . Example 3

[0058] Conduct immune protection experiments with the recombinant vaccine candidate FX1701-CAP 3.1 Immunization and challenge of pigs with FX1701-CAP Ten four-week-old piglets were selected from a farm negative for key pathogens, including PRRSV-2, PRV (pseudorabies virus), PCV (circovirus), and PEDV (porcine epidemic diarrhea virus). Five piglets were inoculated with 3 mL of FX1701-CAP virus containing 10⁵ TCID⁵. Five additional piglets were inoculated with 5 mL of DMEM medium as a negative control. Immunization was by intramuscular injection and intranasal drip.

[0059] Forty-two days after immunization, when viremia had completely disappeared, the pigs were challenged with 10^5 TCID50 of the NADC30-like and PRV2 wild-type viruses in 3 mL of solution via nasal drops and intramuscular injection.

[0060] 3.2 Protection Effect of FX1701-CAP Pig sera were collected at 0, 7, 14, 21, 28, 35 and 42 days after infection (dpi) and tested by Elisa. The IDEXX commercial Elisa kit was used to test the expression level of antibodies against PRRSV and PRV in piglets. The results are shown in Figure 9 .

[0061] Through clinical observation and antibody level testing of piglets, it can be proved that the vaccine candidate strain FX1701-CAP can provide good immune protection for piglets and is safe and reliable.

Claims

1. A recombinant PRRSV strain expressing the porcine circovirus Cap protein, named FX1701-CAP strain, is characterized in that the recombinant PRRSV strain simultaneously contains the HP-PRRSV of lineage 8 (30 amino acid deletion), the NADC30-Like PRRSV of lineage 1 (131 amino acid deletion), and the porcine circovirus type 2 Cap protein.

2. The recombinant PRRSV strain expressing porcine circovirus Cap protein described in claim 1, wherein the specific steps of the construction method are as follows: (1) Extract the PRRSV 1501 strain virus liquid, obtain the full-length viral cDNA by reverse transcription, and use specific primers for PCR amplification to obtain the gene fragment of the 1501 strain; (2) Treat the gene fragments with PacⅠ, BstBI, AfⅡ, AscⅠ, and MluⅠ endonucleases, connect the gene fragments into full-length genomic fragments through enzyme digestion and homologous recombination, and insert them into the PSKⅡ vector; (3) Use EcoRI single enzyme digestion to verify the plasmid and verify the full length of the plasmid to ensure the correct length of the plasmid; (4) The Cap protein gene of porcine circovirus type 2 was amplified using specific primers and inserted between ORF1a and ORF2b of the PRRSV reverse genetic vector by homologous recombination to obtain a recombinant plasmid. The length and correctness of the inserted sequence were verified by enzyme digestion.

3. The method for constructing a recombinant PRRSV strain expressing porcine circovirus Cap protein according to claim 2, wherein the primer sequences used in steps (1) to (4) are as follows: 。 4. The recombinant PRRSV strain expressing porcine circovirus Cap protein according to claim 1 has application value in preparing a vaccine for providing multiple protection against different lineages of PRRSV and porcine circovirus type 2; the multiple protection is intended to provide a new candidate vaccine strain for preventing and controlling mixed infections of different lineages of PRRSV and PCV2.

Images